This invention relates to the crystallization and structure determination of thymidylate kinase (TMK) from Staphylococcus aureus.
Thymidylate kinase (TMK) catalyzes the synthesis of (deoxy)thymidine diphosphate (dTDP) from (deoxy)thymidine monophosphate (dTMP) and ATP along the pathway leading to the synthesis of (deoxy)thymidine triphosphate (dTTP) necessary for DNA synthesis (FIG. 1). Since the phosphorylation of dTDP to dTTP is conducted by a nonspecific diphosphate kinase, TMK is a key player in the regulation of DNA synthesis and is a potential antibacterial target. Interest in thymidylate kinase biochemistry increased when it was recently discovered that this enzyme serves as one of the activators for the AIDS drug, 3xe2x80x2-azido-3xe2x80x2-deoxythymidine (AZT) (L. W. Frick et al., Biochem. Biophys. Res. Comm. 154:124-9 (1988); A. Fridland et al., Mol. Pharmacol. 37:665-70 (1990)). Activation of AZT to azidothymidine triphosphate (AZT-TP) proceeds along cellular phosphorylation pathways to produce the species which is incorporated into growing DNA chains by HIV reverse transcriptase. Similar to its role in serving as a control point for the production of dTTP, thymidylate kinase catalyzes the rate limiting phosphorylation of AZT-monophosphate to AZT-diphosphate (AZT-DP). AZT-DP phosphorylation to AZT-TP is then catalyzed by a nonspecific diphosphate kinase.
In one aspect, the present invention provides a method for crystallizing an S. aureus thymidylate kinase molecule or molecular complex that includes preparing purified S. aureus thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml and crystallizing S. aureus thymidylate kinase from a solution including about 5 wt. % to about 50 wt. % PEG (preferably having a number average molecular weight between about 200 and about 20,000), about 0.05 M to about 0.5 M MgCl2, and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7. In another aspect, the present invention provides a method for crystallizing an S. aureus thymidylate kinase molecule or molecular complex that includes preparing purified S. aureus thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml and crystallizing S. aureus thymidylate kinase from a solution including about 2 mM to about 20 mM xcex2,xcex3-difluoromethylene-bisphosphonate adenosine monophosphate and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7
In another aspect, the present invention provides crystalline forms of an S. aureus thymidylate kinase molecule. In one embodiment, a crystal of S. aureus thymidylate kinase is provided having the trigonal space group symmetry P21.
In another aspect, the present invention provides a scalable three dimensional configuration of points derived from structure coordinates of at least a portion of an S. aureus thymidylate kinase molecule or molecular complex. In one embodiment, the scalable three dimensional set of points is derived from structure coordinates of at least the backbone atoms of the amino acids representing a TMP and/or TMP/ATP substrate binding pocket of an S. aureus thymidylate kinase molecule or molecular complex. In another embodiment, the scalable three dimensional configuration of points is derived from structure coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to an S. aureus thymidylate kinase molecule or molecular complex. On a molecular scale, the configuration of points derived from a homologous molecule or molecular complex have a root mean square deviation of less than about 2.1 xc3x85 from the structure coordinates of the molecule or complex
In another aspect, the present invention provides a molecule or molecular complex that includes at least a portion of an S. aureus thymidylate kinase TMP and/or TMP/ATP substrate binding pocket. In one embodiment, the S. aureus thymidylate kinase TMP substrate binding pocket includes the amino acids listed in Table 1, preferably the amino acids listed in Table 2, and more preferably the amino acids listed in Table 3, the substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 xc3x85, preferably less than about 1.5 xc3x85, more preferably less than about 1.0 xc3x85, and most preferably less than about 0.5 xc3x85 from points representing the backbone atoms of the amino acids. In another embodiment, the S. aureus thymidylate kinase TMP/ATP substrate binding pocket includes the amino acids listed in Table 4, preferably the amino acids listed in Table 5, and more preferably the amino acids listed in Table 6, the substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 xc3x85, preferably less than about 1.5 xc3x85, more preferably less than about 1.0 xc3x85, and most preferably less than about 0.5 xc3x85 from points representing the backbone atoms of the amino acids.
In another aspect, the present invention provides molecules or molecular complexes that are structurally homologous to an S. aureus thymidylate kinase molecule or molecular complex.
In another aspect, the present invention provides a machine readable storage medium including the structure coordinates of all or a portion of an S. aureus thymidylate kinase molecule, molecular complex, a structurally homologous molecule or complex, including structurally equivalent structures, as defined herein, particularly a substrate binding pocket thereof, or a similarly shaped homologous substrate binding pocket. A storage medium encoded with these data is capable of displaying on a computer screen, or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises a substrate binding pocket or a similarly shaped homologous substrate binding pocket.
In another aspect, the present invention provides a method for identifying inhibitors, ligands, and the like for an S. aureus thymidylate kinase molecule by providing the coordinates of a molecule of S. aureus thymidylate kinase to a computerized modeling system; identifying chemical entities that are likely to bind to or interfere with the molecule (e.g., screening a small molecule library); and, optionally, procuring or synthesizing and assaying the compounds or analogues derived therefrom for bioactivity. In another aspect, the present invention provides methods for designing inhibitors, ligands, and the like by providing the coordinates of a molecule of S. aureus thymidylate kinase to a computerized modeling system; designing a chemical entity that is likely to bind to or interfere with the molecule; and optionally synthesizing the chemical entity and assaying the chemical entity for bioactivity. In another aspect, the present invention provides inhibitors and ligands designed or identified by the above methods. In one embodiment, a composition is provided that includes an inhibitor or ligand designed or identified by the above method. In another embodiment, the composition is a pharmaceutical composition.
In another aspect, the present invention provides a method involving molecular replacement to obtain structural information about a molecule or molecular complex of unknown structure. The method includes crystallizing the molecule or molecular complex, generating an x-ray diffraction pattern from the crystallized molecule or molecular complex, and applying at least a portion of the structure coordinates set forth in FIG. 2 to the x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex.
In another aspect, the present invention provides a method for homology modeling an S. aureus thymidylate kinase homolog.
Two crystallographic data sets (with structure factors F) are considered isomorphous if, after scaling,             Δ      ⁢              xe2x80x83            ⁢      F        F    =            ∑              "LeftBracketingBar"                              F            1                    -                      F            2                          "RightBracketingBar"                    ∑              F        1            
is less than about 35% for the reflections between 8 xc3x85 and 4 xc3x85.
The following abbreviations are used throughout this disclosure:
Staphylococcus aureus (S. aureus).
Thymidylate kinase (T. kinase or TMK).
Thymidine 5xe2x80x2-monophosphate (TMP).
Thymidine 5xe2x80x2-diphosphate (TDP).
Thymidine 5xe2x80x2-triphosphate (TTP).
Phospho(enol)pyruvate (PEP)
Reduced nicotinamide adenine dinucleotide (NADH)
Oxidized nicotinamide adenine dinucleotide (NAD+)
Pyruvate kinase (PK)
Lactate dehydrogenase (LDH)
Nucleoside-5xe2x80x2-diphosphate kinase (NDP-Kinase)
(Deoxy)thymidine monophosphate (dTMP).
(Deoxy)thymidine diphosphate (dTDP).
(Deoxy)thymidine triphosphate (dTTP).
Adenosine 5xe2x80x2-diphosphate (ADP).
Adenosine 5xe2x80x2-triphosphate (ATP).
Isopropylthio-xcex2-D-galactoside (IPTG).
Dithiothreitol (DTT).
Dimethyl sulfoxide (DMSO).
Polyethylene glycol (PEG).
Multiple anomalous dispersion (MAD).
The following amino acid abbreviations are used throughout this disclosure: